1. Field of the Invention
The present invention relates to a method for detecting a cancer cell characterized by detecting an expression product of a gene capable of changing an expression level thereof owing to canceration. The present invention relates to a gene capable of changing an expression level thereof and a product of the gene owing to canceration.
2. Discussion of the Related Art
Cancers constitute the top of the causes for mortality in Japan since 1981, and a gastric cancer occurs especially at the highest frequency. Recently, there has been known that there is a multi-stage carcinogenic mechanism in the course from a normal cell to a cancer cell [Fearon, E. R. et al., Cell, 61, 759-767 (1990); Sugimura, T., Science, 258, 603-607 (1992)] for which the accumulation of the abnormality in a plurality of genes including DNA repair genes, tumor suppressor genes and oncogenes is essential. Generally, the instability of a gene and the inactivation of a tumor suppressor gene are involved in the development of a cancer, and the activation of an oncogene and/or the overexpression of a growth factor are involved in the advancement and malignancy of a cancer.
The instability of a gene includes the instability of gene associated with abnormality in a DNA mismatch repair system and the instability at a chromosomal level. An example of the former includes the difference in the chain length of a simple repeated sequence present in a genome between a cancer site and a non-cancer site in the same individual (microsatellite instability) [Thibodeau, S. N. et al., Science, 260, 816-819 (1993)], and an example of the latter includes an interchromosomal translocation. The interchromosomal translocation may cause to express a protein which is not found in normal cells, or the interchromosomal translocation may affect an expression level of a protein, even if it is expressed in normal cells. In fact, in human chronic myelocytic leukemia, bcr gene is fused with c-abl gene by the interchromosomal translocation, and there has been confirmed an expression of a hybrid mRNA transcribed from bcr-abl fusion gene, which is absent in normal cells. Further, there has been confirmed that an introduction of bcr-abl fusion gene into an animal results in an onset of leukemia [Watson, J. D. et al., Molecular Biology of Recombinant DNAS, 2nd Ed., Maruzen K. K., 309 (1992)].
The inactivation of a tumor suppressor gene includes, for example, an inactivation of p53 gene. The inactivation is considered to be caused by a deletion within the gene, or a point mutation occurring in a particular portion of an encoding region [Nigro, J. M. et al., Nature, 342, 705-708 (1989); Malkin, D. et al., Science, 250, 1233-1238 (1990)]. In addition, since the deletion and the point mutation of the p53 gene are observed in various cancers, and are as frequent as 60% or higher especially in cases of a gastric cancer at an early stage [Yokozaki, H. et al., Journal of Cancer Research and Clinical Oncology, 119, 67-70 (1992)], the detection of these mutations is considered to be useful for detecting a cancer at an early stage.
On the other hand, p16/MTS1 gene has been known to be a gene which is inactivated owing to a homologous deletion, and high-frequency homologous deletions have been observed in cases of a glioma, a pancreatic cancer and a urinary bladder cancer [Cairns, P. et al., Nature Genetics, 11, 210-212 (1995)]. p16 Protein regulates a cell cycle, and the abnormality in p16 expression has been suggested to be involved in the canceration of a cell [Okamoto, A. et al., Proceedings of the National Academy of Sciences of the United States of America, 91, 11045-11049 (1994)].
As the causation for the activation of an oncogene, there can be included, for example, a viral insertion mutation in a proximity of an oncogene and an interchromosomal translocation. For example, a viral insertion mutation has been confirmed in lymphoma of a chicken which is caused by an avian leukosis virus (ALV). In this case, it has been found that DNA of an ALV is inserted in the proximity of a gene c-myc, and, by potent viral enhancer and promoter, a normal c-myc is overexpressed, and a new sequence which is different partially from the normal gene has been expressed. In addition, in a certain kind of human B cell tumor, there has been confirmed that c-myc, which is one of oncogenes, is located near a potent transcription signal of immunoglobulin by the interchromosomal translocation, whereby increasing its expression level of the mRNA. In this case, no difference has been found between a protein for c-myc in a cancer cell and a protein for c-myc expressed in a normal cell, and the canceration is considered to be caused by an increase in the expression level of the c-myc mRNA [Watson, J. D. et al., Molecular Biology of Recombinant DNAS, 2nd Ed., Maruzen K. K., 305-308 (1992)].
An overexpression of a growth factor includes, for example, an overexpression of C-Met which encodes a hepatocyte growth factor receptor. There has been confirmed that the abnormality in expression of the C-Met is observed as an expression of mRNA having the length of 6.0 kb which is not found in a normal mucous membrane at an early stage of gastric cancer [Kuniyasu, H. et al., International Journal of Cancer, 55, 72-75 (1993)], or is observed at a high frequency, and that a correlation between the gene amplification and the degree of the cancer malignancy is observed [Kuniyasu, H. et al., Biochemical and Biophysical Research Communications, 189, 227-232 (1992)].
As examples of confirming the correlation between the gene abnormality and the degree of cancer malignancy, in addition to that of the c-Met mentioned above, there have been confirmed that an amplification and/or an overexpression of an oncogene C-erbB2 gene is found in mammary cancers, ovarian cancers, gastric cancers and uterine cancers [Wright, C. et al., Cancer Research, 49, 2087-2090 (1989); Saffari, B. et al., Cancer Research, 55, 5693-5698 (1995)]; and that an amplification and/or an overexpression of an oncogene K-sam gene is found in a poorly-differentiated adenocarcinoma which is one tissue type of gastric cancer [Tahara, E. et al., Gastric Cancer, Tokyo, Springer-Verlag, Published in 1993, 209-217], respectively.
As described above, the information concerning the gene involved in the development and the advancement of a cancer as well as the abnormality of such genes has been increasing, and the genetic diagnosis of a biopsy material may serve for an early diagnosis and an assessment of the degree of malignancy of a cancer. However, since a carcinogenic mechanism comprises multiple steps and requires an accumulation of a plurality of mutations, a large part of the genes associated with the canceration have still yet been unknown, and further study is necessary. Recently, a gene therapy in which a normal p53 gene is introduced into a cancer cell whereby suppressing the proliferation of the cancer cell is now at a stage of a clinical trial. Therefore, the solution for a cancer-suppressing gene can shed light not only in the diagnosis but also in the gene therapy.
Accordingly, a first object of the present invention is to provide a method for detecting cancerated cell and a method for determining a degree of malignancy, on the basis of finding a gene usable as an index for carcinogenesis, particularly a gene capable of changing expression conditions thereof by canceration of a cell, and measuring an expression level of the gene in a resected specimen. A second object of the present invention is to provide a kit used for the above method for detecting a cancer cell and/or a method for determining a degree of malignancy of the cell. A third object of the present invention is to provide a method for controlling proliferation of a cancer cell by using a substance specifically binding to a gene capable of serving as an index for carcinogenesis or an expression product of the gene. Furthermore, a fourth object of the present invention is to provide a novel peptide associated with canceration, and a nucleic acid encoding the peptide. These and other objects of the present invention will be apparent from the following description.
To summarize the present invention, a first invention of the present invention is an invention pertaining to a method for detecting a cancer cell in a resected specimen, characterized by determining a change in an expression level of a gene selected from genes of which cDNA corresponds to a DNA comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 1 to 16 and 66 to 68 in Sequence Listing, or a DNA capable of hybridizing with a nucleic acid as shown in any one of SEQ ID NOs: 1 to 16 and 66 to 68 in Sequence Listing under stringent conditions by, for example, determining a change of an expression level of mRNA or a change of a protein expression level.
A second invention of the present invention is an invention pertaining to a kit for detecting cancer by the method for detecting of the present invention, characterized in that the kit comprises as an essential constituent any one of primers for amplifying mRNA as an index for a change in an expression level, a probe capable of hybridizing with the above mRNA, or an antibody recognizing a protein as an index for the change in expression level.
A third invention of the present invention is a method for controlling proliferation of a cancer cell by using a substance specifically binding to the gene or an expression product thereof, characterized in that cDNA of the gene corresponds to a DNA comprising a nucleotide sequence any one of sequences of SEQ ID NOs: 1 to 16 and 66 to 68 in Sequence Listing, or a DNA capable of hybridizing with DNA as shown in any one of sequences of SEQ ID NOs: 1 to 16 and 66 to 68 in Sequence Listing, wherein the method gives transcriptional control of the gene and/or functional control of an expression product thereof, and the like.
A fourth invention of the present invention is an invention pertaining to a peptide usable for detecting cancer and a nucleic acid encoding the peptide, characterized in that the peptide consists of an amino acid sequence comprising an entire portion of an amino acid sequence as shown in any one of SEQ ID NOs: 17 to 19, 69 and 70 in Sequence Listing or a partial portion thereof and a nucleic acid encoding the peptide.
A fifth invention of the present invention pertains to an antibody usable for detecting cancer, the antibody recognizing the above peptide of the fourth invention.
Incidentally, the term xe2x80x9cresected specimenxe2x80x9d used in the present specification refers to blood, urine, feces, tissue resected by surgery, and the like. On the other hand, the term xe2x80x9ccancer-associated genexe2x80x9d refers to a gene in which the expression conditions thereof change with canceration of a cell.
In order to achieve the objects mentioned above, the present inventors have found a cancer-associated gene by comparing the intracellular expression levels of genes between a cancer tissue and a control normal tissue of a cancer patient, and they have found that cancer cells can be detected by comparing the expression level of this gene. In addition, they have found a novel gene in this cancer-associated gene, whereby completing the present invention.
The terms xe2x80x9ccancer tissuexe2x80x9d and xe2x80x9ccontrol normal tissuexe2x80x9d used in the present specification mean a tissue constituting a region of cancerous lesion in a multicellular individual and a tissue constituting a region which is identical spatially to the cancer tissue in the same individual but functions normally.